Antitumoral composition based on polypeptides having human interleukin 2 activity

ABSTRACT

The use of a polypeptide having human interleukin 2 activity for preparing a pharmaceutical composition for treating malignant epithelial tumours of the upper respiratory/alimentary tract or the thymus, is described.

The present invention relates to the use of a polypeptide having theactivity of human interleukin 2 for the preparation of pharmaceuticalcompositions intended for the treatment of epithelial malignant tumours.

Interleukin 2 (IL₂) which is a lymphokine produced by activated Tlymphocytes possesses an immunomodulatory activity and an anti-tumouractivity described for example by Fletcher, M et al. (LymphokineResearch 6 1987 47-57), which activities include in particular theability to initiate the proliferation of the T lymphocyctes and theinduction of the cytotoxicity of the NK (natural killer) cells and theLAK (lymphokine activated killer) cells. It has been observed that theadministration of IL₂ either on its own at a high dose or combined withLAK cells is able to induce the regression of certain cancers present ina mouse and in patients having metastatic cancers such as melanoma,cancer of the kidney, colorectal cancer or non-Hodgkin's lymphoma(Rosenberg, S A et al. N. Engl. J. Med. 1987 316 889-897).

Malignant epithelial tumours, or epitheliomas, which are tumours causedby a neoplasic proliferation of the epithelial cells or epidermis, orepidermoid mucous membranes, are histologically diagnosed, in particularin the upper aerodigestive tracts or in the thymus.

The epitheliomas of the upper aerodigestive tracts concern the varioussites of the pharynx, amongst which the nasopharynx which represents arelatively frequent localization with different histological sub-types,most often of lymphoepithelial type characterized by an undifferentiatedepithelioma infiltrated by lymphocytes.

The most important treatment of epitheliomas is radiotherapy, generallypreceded by polychemotherapy or combined with the latter. The prognosis,variable according to the development of the epithelioma, isunfavourable in as much as a large percentage of patients treateddevelop metastases afterwards. Thus a study involving 49 patientssuffering from carcinomas of the nasopharynx and treated byradio-therapy, either on its own or preceded by chemotherapy, describesan average survival rate of 42% after 5 years (Stein, M et al. J. Surg.Oncol. 1988 37 (2) 84-88). In spite of a prognosis which seems betterwhen lymphoepitheliomas are concerned, a low survival rate of only 13%is observed after 5 years in a study involving 150 patients sufferingfrom nasopharyngeal carcinoma, most frequently of lymphoepithelial type,although a complete remission rate of about 65% was described afterradiotherapy preceded or not preceded by chemotherapy (Koppibar, S Betal. J. Surg. Oncol. 1988 39 (3) 179-182). The absence of an effectivetreatment is also recognized, notably in 29 children all suffering fromlymphoepitheliomas of the nasopharynx, amongst which more than 50%developed secondary metastases within 2 years once the radiotherapy hadstopped (Pao, W J et al. In. J. Radiat. Oncol. Biol. Phys 1989 17 (2)299-305).

Malignant thymomas which are tumours of the thymus caused by aneoplastic proliferation of the epithelial cells of the thymus includemainly invasive malignant thymomas without atypia, amongst which therecan possibly be singled out the lymphoepithelial or lymphocytarysub-type according to the cell population which accompanies them, andthe atypical carcinomas of the thymus.

In contrast to benign encapsulated thymomas for which suitabletreatments exist such as surgery, radiotherapy, alone or combined,malignant thymomas, in particular invasive malignant thymomas withoutatypia, are not very sensitive to conventional treatments. Furthermore,the frequency with which they appear is increasing (23 to 66% of allthymomas).

Thus invasive malignant thymomas treated either by surgery, or byradiotherapy as the first treatment, followed optionally bychemotherapy, have an unfavourable prognosis: up to 30% of the patientssecondarily develop metastases. The results described for patientssuffering from lymphoepithelial thymomas (Arriagada, R et al. Eur JCancer Clin Oncol 20 (No. 1) 69-74 1984) or for patients suffering fromlympho-epithelial or lymphocytary thymomas (Goldel, N et al. Cancer 631493-1500 Apr. 15 1989) are deceptive with the exception of a fewencouraging results described after treatment by polychemotherapy ofinvasive malignant thymomas rich in undifferentiated epithelial cells(Dy, C et al. Journal of Clinical Oncology 6 (No. 3) 536-542 March1988). Therefore no recognized treatment exists, in particular for theinvasive forms of malignant thymomas or for atypical carcinomas of thethymus for which high rates of resistant cases are observed, inparticular to chemotherapy.

No clinical result has described the use of IL₂ in the treatment ofepithelial tumours of the upper aerodisgestive tracts.

A clinical result has shown the ineffectiveness of the therapy inmalignant thymomas with IL₂ on its own, notably with a mutein of IL₂ ina patient suffering from thymoma having received a dose of 1×10⁶ U/M² byintravenous route perfused over 6 hours according to a cycle constitutedby 2 times 5 days (Kolitz, J E et al. Journal of Biological ResponseModifiers 6 412-429 1987).

Now in a surprising way, the Applicant has just obtained results showingthat IL₂ on its own has an activity on epitheliomas.

Therefore the invention relates to the use of a polypeptide having theactivity of human interleukin 2 for preparing a pharmaceuticalcomposition intended for the treatment of epithelial malignant tumoursof the upper aerodigestive tracts or of the thymus.

By polypeptide having the activity of human IL₂ is meant natural humanIL₂, recombinant human IL₂, that is obtained by the technology ofrecombinant DNA, for example such as that described by Taniguchi, T etal. Nature 1983 302 305-310 or in the Patent EP 91530 B, alleles orderivatives of these products as described for example by Ju, C et al.J. Biol. Chem. 1987 262 5723-5731.

The tumours of the upper aerodigestive routes with which the inventionis concerned include epitheliomas of the pharynx in general, for exampleepitheliomas of the nasopharynx, of the tonsils or of the oesophagus.The tumours of the thymus with which the invention is concerned includeepithelial, lymphoepithelial or lymphocytary invasive malignant thymomaand carcinomas of the thymus.

Notably a subject of the invention is the use characterized in that thetumour of the upper aerodigestive tracts is a lymphoepithelioma,histologically diagnosed.

Also a subject of the invention is the use characterized in that thetumour of the thymus is a lymphoepithelial invasive malignant thymoma,diagnosed by the usual histological, cytological and biologicalexaminations. The use according to the invention is intended for thetreatment of malignant thymoma, for the treatment of the recurrence ofthe latter or for its preventative treatment.

The use of IL₂ according to the invention is illustrated by a partialresponse in a patient suffering from malignant thymoma having undergone,without success, a previous chemotherapy treatment.

A particular subject of the invention is the use characterized in thatthe human IL₂ is a pure recombinant IL₂.

The pharmaceutical compositions prepared according to the inventioncontain a recombinant human IL₂, alleles or derivatives of the latter,as described above, for which purification techniques known to a manskilled in the art are used, which allow the preparation of pureproducts.

A more particular subject of the invention is the use characterized inthat the IL₂ is a non-glycosylated recombinant IL₂ in reduced form. Thenon-glycosylated IL₂ used is notably that having the sequence of naturalIL₂ with 133 aminoacids, optionally with an additional N-terminalmethionin, of which the 3 cysteines in position 58, 105 and 125 are inreduced form, showing a biological activity comparable to that ofoxidized IL₂ having the same sequence containing a disulphide bridge inposition 58-105 and which is described in the European PatentApplication EP 0353150. By reduced form is meant that the cysteineremainders contained by the IL₂ comprise a free sulphhydryl group thedetermination of which is made, for example, by spectrophotometry withdithiodipyridine as the reagent of the thiols. The biological activityis determined by measuring the proliferation of leukemic cell lines ofIL₂ CTLL-2 dependent mice, with a colorimetric test using a tetrazoliumsalt (Mossman, T J Immunol. Meth 1983 65 55-63). The specific activityof the recombinant IL₂ 's used in the invention is at least equal to0.5×10⁷ U BRMP/mg, preferably 1×10⁷ U BRMP/mg. The IL₂ activity unit isdefined as the quantity which produces 50% of the maximum response inthe test. A "Biological Response Modifier Program" (BRMP), referenceagent human IL₂ "jurkat" sample provided by the National CancerInstitute (NCI) is used as standard.

A subject of the invention is especially the use characterized in thatIL₂ is administered by intravenous route as a continuous perfusion at adose of 2 to 25×10⁶ U/M² per day and more especially that characterizedin that IL₂ is administered at a dose of 20×10⁶ U/M² per day.

A subject of the invention is quite especially the use characterized inthat IL₂ is administered in a cycle of 3 to 5 consecutive days andcharacterized in that IL₂ is administered repeatedly for at least 3non-consecutive cycles.

The dose administered, the frequency of the injection and the durationof the treatment vary as a function of the condition of the patient.

The IL₂ is contained in a pharmaceutical composition, preferablylyophilized in a dosage bottle containing 0.05 to 2 mg of activeingredient and which is reconstituted with distilled water forinjection. The solution obtained is immediately diluted with a solute,for example 5% glucose, for administration as an intravenous perfusion.

According to the preferred use of the invention, the IL₂ is the reducedrecombinant IL₂ above, an example of the pharmaceutical preparation ofwhich is given further on, the dose is 20×10⁶ U/M² per day, according toa cycle of 3 to 5 consecutive days, the duration of administration is 3non-consecutive cycles as a continuous perfusion by intravenous route.

All the publications which are mentioned are incorporated in the text ofthe present Application for reference.

The following examples illustrate the invention without however limitingit:

EXAMPLE 1 Pharmaceutical Composition for Perfusion

A preparation for injection by intravenous route as a perfusion is madeof formula:

    ______________________________________                                        reduced IL.sub.2       0.5   mg                                               citric acid            5     mg                                               mannitol               50    mg                                               sterilized water       1     ml                                               5% glucose             50    ml                                               ______________________________________                                    

EXAMPLE 2 Clinical Study in the Treatment of a Malignant Thymoma

The study includes a patient having a lymphoepithelial malignant thymomahistologically confirmed invading the mediastinum, the right lung, thepleura, the pericardium and the diaphragm, having received a firstcourse of treatment with 2 cycles of conventional polychemotherapycomprising cisplatin, endoxan and vindesine, then a second course oftreatment with a high dose (100 mg/M²) of adriamycin without aregression of the tumour having been observed.

The absence, under chemotherapy, of a regression of the mediastinal andpleuropulmonary masses shown by radiography and the thorax scanner, leadto the initiation of a treatment by IL₂ four and a half months afterthis chemotherapy was completed.

The compositions of IL₂ prepared according to the invention permit adose of 20×10⁶ U/M² per day to be injected as a continuous perfusion byintravenous route for 3 to 5 consecutive days according to a first cycleof 5 days, a second cycle of 4 days and a third cycle of 3 days with aninterval of 9 days between each cycle. The tumourous lesions of thepatient are evaluated before and after each cycle by radiography then byscanning. The compositions described in Example 1 are used.

The treatment with IL₂ brought about a partial response at the level ofthe pleural masses which decreased by more than 75%, observed byradiography and confirmed by a thorax scanner, without modification ofthe thymic mass. A thoracotomy is carried out during the course of thetreatment with IL₂ to check the condition of the thymic sheath. Theresidual tumourous mass can be completely removed by a surgeon and isfound to be massively necrotic whereas the complete removal of theresidual metastases at a pulmonary and diaphragmatic level is nottechnically possible.

We claim:
 1. A method of treating an epithelial malignant tumor of the upper aerodigestive tract or of the thymus of a warm-blooded animal comprising administering to said warm-blooded animal an antitumorally effective amount of interleukin
 2. 2. The method of claim 1 wherein the tumor is a lymphoepithelioman.
 3. The method of claim 1 wherein the tumor is a lymphoepithelial invasive malignant thymona.
 4. The method of claim 1 wherein the interleukin 2 is a pure recombinant IL-2.
 5. The method of claim 4 wherein the interleukin 2 is non-glycosylated recombinant IL-2 in reduced form.
 6. The method of claim 5 wherein the interleukin 2 is administered intravenously by continuous perfusion at a dose of 2 to 25×10⁶ U/M² per day.
 7. The method of claim 6 wherein the dose is 20×10⁶ U/M² per day.
 8. The method of claim 7 wherein the administration is in a cycle of 3 to 5 consecutive days.
 9. The method of claim 8 wherein the administration is repeated for at least 3 non-consecutive days. 